The Contribution Of The Polymerase Chain Reaction Biology Essay

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Food poisoning is an illness caused by eating food that has been contaminated. Food borne pathogens have been recognised as one of the most common sources causing food poisoning worldwide and in severe cases even death. Typical symptoms of food poisoning would include nausea, vomiting, stomach cramps, and diarrhoea within 48 hours of consuming a contaminated food or drink. Depending on the pathogen that contaminated the food the patient may also experience fever and chills, hematochezia, dehydration and nervous system damage may follow. Foods most likely to get contaminated are meats, poultry, ready to eat foods and dairy products such as milk, cheese and eggs. They can become contaminated at any stage during growth, processing or cooking. Food poisoning is generally caused by foods consumed that either hasn't been cooked to the right temperature for the correct length of time, foods that have not been chilled to the necessary temperature to stop bacterial growth, foods that have been handled by unwashed hands, foods that are passed their "use by" dates and cross contamination.

Food poisoning can be divided into two categories: infectious and toxic agents. Infectious agents include viruses, bacteria and parasites. Toxic agents include poisonous mushrooms, improperly prepared exotic foods or pesticides on fresh produce. A common type of bacteria that contaminate food would be campylobacter, a gram negative bacterium which is viable but not culturable, is one of the most common causes of food poisoning in England. They are usually found in raw meat and poultry, unpasteurised milk and untreated water. Salmonella, first extracted from a bovine source in Germany, is now a very common food borne pathogen found in raw meat, poultry and dairy products. Escherichia coli are bacteria found in the normal flora of the gut of many animals are completely harmless. However the strain E.coli serotype 0157:H7 can cause serious food poisoning. They are found in undercooked beef and unpasteurised milk. Another common cause of food poisoning would be a group known as viruses. There are two common types; the rotavirus and the norovirus. Rotavirus are more common in children than adults as people generally build an immune response to these with age, whereas norovirus can affect people of any ages. Other food poisoning pathogens could be parasites and toxins found in foods such as contaminated fish.

Microbiological assessment is important to determine the safety and quality of food. In the past detection and identification of microorganisms (in foods, animal faeces and environmental samples) have relied mainly on cultural techniques. These methods are the most reliable and accurate in the detection of food borne pathogens. However they are labour intensive, have long processing times and are costly. Conventional methods consist of blending the sample with a selective enrichment medium to increase the population of the target organism, then plating this sample onto selective or differential agar plates to isolate pure cultures. These cultures are then examined by phenotypic analysis or metabolic fingerprinting. A major disadvantage to this method is that it can take 2-3 days for any results to show up and up to 7-10 days for confirmation. Advances in technology lead to development of a Stomacher for sample processing which is a paddle blender that ensures that the whole sample is immersed within the culture medium. Another advance in the technique would be improved liquid and selective agar media, instruments for plating and counting bacteria, and commercial identification kits. To avoid delay, many of the modern methods use a conventional method along with an automated or semi-automated DNA, antibody, or biochemical based method. This allows the confirmation of the bacteria in 3-4 days.

Traditional methods of identification of food borne pathogens, which cause disease in humans, were time consuming and laborious, so there was a need for the development of more innovative methods for the rapid identification of food borne pathogens (Naravaneni, Jamil 2004). Advances in biotechnology led to the development of rapid methods that minimize manipulation, provide results in less time, and reduce costs. These methods generally include immune-based and DNA based assays. Immunological or antibody-based assays include enzyme linked-immunosorbent assays (ELISA) and immune-chromatographic or "dipstick" assays. Genetic methods include polymerase chain reaction (PCR), DNA hybridisation and DNA microarrays.

The basic principle of immunoassay detection is the binding of the antibodies to a target antigen, followed by the detection of the antigen-antibody complex. Antibodies are produced by the body in response to a specific invading pathogen i.e. the one causing the food poisoning. The most important characteristic of an antibody is its ability to recognise only the target antigen in the presence of other organisms and interfering food components. In addition, the successful use of antibodies to detect pathogens depends on the stable expression of target antigens in a pathogen, which are often influenced by temperature, preservatives, acids, salts or other chemicals found in foods.

PCR (polymerase chain reaction) is a powerful technique that has revolutionised molecular biology research and has application in the diagnosis of microbial infections and genetic diseases, as well as in detection of pathogens in food, faecal, and environmental samples. It has become the most frequently used method for amplifying DNA since it was discovered by Millus. PCR is an in vitro method that employs a heat stable DNA polymerase enzyme, for example Taq polymerase, a template DNA from the pathogens being detected, and two complimentary oligonucleotide primers that are designed to amplify a specific region on the template DNA. The choice of DNA region to be amplifies determines the specificity of detection. Suitable targets for detecting food borne pathogens include ribosomal RNA genes and protein genes. Assays based on the PCR are rapid methods in confirming the presence or absence of specific pathogens in foods. A typical amplification needs to be 20-40 cycles, which amplifies specific pieces of template DNA at more than a billion-fold. Before PCR can take place the PCR has to be separated from other substances given in a sample, this step is called the pre-PCR treatment.

A common method used to separate DNA from the other substances in the sample would be immunomagnetic separation. The technique has been proven to be efficient for separating certain eukaryotic cells from fluids and heterogeneous samples such as blood, food and faecal samples. Paramagnetic beads coated with antibodies to surface antigens of bacteria are used to separate and extract concentrated samples of organisms from the sample. Bacteria that bind to the beads for aggregates which are drawn to the side of the test tube by a magnet and inhibitory factors in the test sample can be removed by changing the medium. The bacteria that are attached to the beads are lysed releasing their DNA into the supernatant which can then be used for PCR. (Enroth, Engstrand, 1995)

Each amplification cycle of a PCR consists of a heat denaturation phase where the strands are heated to about 91°c and as a result single strands are generated from a double stranded DNA. Due to the fact that the hydrogen bonds that occur between the complementary nucleotide base pairs are weak, they break at high temperatures; whereas the bonds between the deoxyribose and the phosphate are stronger covalent bonds the single strands remain intact. The second step is the annealing phase when the two primers bind to the complementary single-stranded target sequence produced during the denaturing process. To ensure that the primers anneal to the target sequence with high specificity, annealing usually occurs between 40°c-65°c. The final step is the extension phase when the DNA polymerase synthesises a strand that is complementary to the template at around 72°c using dNTP's. The synthesis is carried out from the 3' end of the primer to the 5' to 3' direction. This approach has been frequently used for a growing number of studies to detect and characterise food borne pathogens.

After amplification the products of PCR have to be detected. The detection is carried out by running separating the products from PCR on agarose gel electrophoresis to be separated by size a dye would be used in order to detect the bands that are formed. A step also had to be taken for amplicon confirmation. This can be done for example by hybridisation with a DNA probe, combining the PCR with a hybridisation step enhances the assays sensitivity and specificity.

Real-time PCR or quantitative PCR is a technique that enables both the detection and quantification of one or more specific sequences in a sample of DNA. This procedure also follows the general principle of PCR, however the amplified DNA is detected as the reaction progresses in real time, which is different compared to standard PCR, where the product of the reaction is detected at the end. Real-time PCR is based on the detection of the fluorescence produced by a reporter molecule which intensifies as the reaction proceeds. This occurs due to the accumulation of the PCR product with each cycle of amplification. These fluorescent reporter molecules include non-specific fluorescent dyes such as SYBR® Green that intercalate with the double stranded DNA, or sequence of specific probes. They consisting of oligoucleotides that are labelled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary DNA target. Common probes used are the TaqMan probes.

TaqMan probes are used to increase the specificity of real-time PCR assays. The probe relies on the 5'-3' exonuclease activity of the Taq polymerase to cleave a dual labelled probe during hybridisation to the complementary target sequence and flurophore-based detection. In real-time PCR the fluorescence allows quantitative measurements of the accumulated product during the exponential stages of the PCR. The TaqMan probe increases the specificity of the detection. They consist of a fluorophore covalently attached to the 5'-end of the oligonuclotide probe and a quencher at the 3'-end. The quencher molecule quenches the fluorescence emitted by the flourophore. When the target strand is amplified in the PCR reaction, the Taq polymerase will degrade the prbe resulting in separation of the fluorophore and the quencher, hence the fluorescence of the sample will be proportional to the number of amplicons that have been created. (Baric et al, 2006)

Real-time PCR can also be combined with reverse transcription. Reverse transcription PCR is a modified method of PCR in which RNA is used instead of DNA as the initial template. In contrast to the detection of DNA from the pathogens that would be done in normal PCR, the detection of cDNA from mRNA encoded by a pathogen. Reverse-transcription PCR can be used to detect active cells.

Multiplex real-time PCR, for example quadruplex real-time PCR is an advantage against the culturing methods as you can use numerous amounts of selective DNA in one PCR. Multiplex real-time PCR can be used to detect many different target genes in a single reaction tube simultaneously. Recent reports have shown that multiplex real-time PCR greatly improves specificity and sensitivity for the detection of pathogens, in particular V.cholerae through either melting curve analysis or using different fluorophore labelled probes. (Haung, 2009)

When comparing conventional PCR with real-time PCR one would say, based on evidence and research carried out, that real-time PCR is the better method out of the two. As stated in research carried out by Huang et al, 2009; Real-time PCR allows the detection of amplification product accumulation through fluorescence intensity changes in a closed-tube setting, which is faster and more sensitive than conventional PCR (Huang et al, 2009). They also state that an advantage of real-time PCR is that it can simultaneously be used to serogroup the pathogen and also determine its toxin status. A comparison was carried out between real-time PCR, conventional PCR and different staining techniques for diagnosing Pneumocystic jiroveci pneumonia from bronchoalveolar lavage specimens by Flori, P et al, 2003. They collected specimens from 150 patients that were evaluated for identification of Pneumocystic jiroveci using real-time PCR, conventional PCR and staining techniques. The results showed that the specificity of real-time PCR in that case was 98.6% in comparison to 87% for conventional PCR respectively. It was stated that the technique was rapid. (Flori et al, 2003). A disadvantage of real-time PCR could ne that it costs slightly more than the conventional mothod due to the high costs of consumables and reagents, however the method is less laborious and therefore the material costs could compensate for the personnel costs. (Baric et al,2006).

Conventional PCR assays need to use gel electrophoresis to separate the products for post-amplification analysis, which has a low throughput, is labour intensive, and is susceptible to carry over contamination. By eliminating the gel electrophoresis step with real-time PCR, the assay reduces the risk of contamination, increases assay throughput and shortens testing time. Therefore the advantage is that in real-time PCR there is no need for post PCR processing which not only saves time, but money and resources too. Using PCR also means that smaller samples can be used to obtain highly specific results. Contributions of PCR techniques have completely revolutionised the approach to the detection of food borne pathogens. Real-time PCR techniques are easy to perform, have high sensitivity, more specificity compared to cultural methods.